The present invention relates to novel classes of compounds which are inhibitors of interleukin-1xcex2 converting enzyme and related proteases (xe2x80x9cICE/ced-3 family of cysteine proteasesxe2x80x9d). This invention also relates to pharmaceutical compositions comprising these compounds and to methods of using such pharmaceutical compositions. The compounds, pharmaceutical compositions and methods of this invention are particularly well suited for inhibiting the protease activity of the ICE/ced-3 family and consequently, may be advantageously used as agents against interleukin-1 (xe2x80x9cIL-1xe2x80x9d) mediated diseases, including inflammatory diseases, autoimmune diseases and neurodegenerative diseases and for inhibiting unwanted apoptosis in various disease states such as ischemic injury to the heart (e.g., myocardial infarction), brain (e.g., stroke), and kidney (e.g., ischemic kidney disease).
Interleukin 1 (xe2x80x9cIL-1xe2x80x9d) is a major pro-inflammatory and immunoregulatory protein that stimulates fibroblast differentiation and proliferation, the production of prostaglandins, collagenase and phospholipase by synovial cells and chondrocytes, basophil and eosinophil degranulation and neutrophil activation. Oppenheim, J. H. et al., Immunology Today, 7:45-56 (1986). As such, it is involved in the pathogenesis of chronic and acute inflammatory and autoimmune diseases. IL-1 is predominantly produced by peripheral blood monocytes as part of the inflammatory response. Mosely, B. S. et al., Proc. Nat. Acad. Sci., 84:4572-4576 (1987); Lonnemann, G. et al., Eur. J. Immunol., 19:1531-1536 (1989).
IL-1xcex2 is synthesized as a biologically inactive precursor, proIL-1xcex2. ProIL-1xcex2 is cleaved by a cysteine protease called interleukin-1xcex2 converting enzyme (xe2x80x9cICExe2x80x9d) between Asp-116 and Ala-117 to produce the biologically active C-terminal fragment found in human serum and synovial fluid. Sleath, P. R. et al., J. Biol. Chem., 265:14526-14528 (1992); A. D. Howard et al., J. Immunol., 147:2964-2969 (1991).
ICE is a cysteine protease localized primarily in monocytes. In addition to promoting the pro-inflammatory and immunoregulatory properties of IL-1xcex2, ICE, and particulary its homologues, also appear to be involved in the regulation of cell death or apoptosis. Yuan, J. et al., Cell, 75:641-652 (1993); Miura, M. et al., Cell, 75:653-660 (1993); Nett-Giordalisi, M. A. et al. J. Cell Biochem., 17B:117 (1993). In particular, ICE or ICE/ced-3 homologues are thought to be associated with the regulation of apoptosis in neurogenerative diseases, such as Alzheimer""s and Parkinson""s disease. Marx, J. and M. Baringa, Science, 259:760-762 (1993); Gagliardini, V. et al., Science, 263:826-828 (1994).
Thus, disease states in which inhibitors of the ICE/ced-3 family of cysteine proteases may be useful as therapeutic agents include: infectious diseases, such as meningitis and salpingitis; septic shock, respiratory diseases; inflammatory conditions, such as arthritis, cholangitis, colitis, encephalitis, endocerolitis, hepatitis, pancreatitis and reperfusion injury, ischemic diseases such as the myocardial infarction, stroke and ischemic kidney disease; immune-based diseases, such as hypersensitivity; auto-immune diseases, such as multiple sclerosis; bone diseases; and certain neurodegenerative diseases, such as Alzheimer""s and Parkinson""s disease.
ICE inhibitors represent a class of compounds useful for the control of the above-listed disease states. Peptide and peptidyl inhibitors of ICE have been described. However, such inhibitors have been typically characterized by undesirable pharmacologic properties, such as poor oral absorption, poor stability and rapid metabolism. Plattner, J. J. and D. W. Norbeck, in Drug Discovery Technologies, C. R. Clark and W. H. Moos, Eds. (Ellis Horwood, Chichester, England, 1990), pp. 92-126. These undesirable properties have hampered their development into effective drugs.
Accordingly, the need exists for compounds that can effectively inhibit the action of the ICE/ced-3 family of proteases, for use as agents for preventing unwanted apoptosis and for treating chronic and acute forms of IL-1 mediated diseases, such as inflammatory, autoimmune or neurodegenerative diseases.
The compounds of this invention incorporate a conformationally constrained dipeptide mimetic. This mimetic exhibits improved properties relative to their peptidic counterparts, for example, such as improved absorption and metabolic stability resulting in enhanced bioavailability.
One aspect of this invention is compounds of the formula: 
wherein:
n is 1 or 2;
m is 1 or 2;
A is R2COxe2x80x94, R3xe2x80x94Oxe2x80x94COxe2x80x94, or R4SO2xe2x80x94;
a group of the formula: 
further wherein:
R1 is a hydrogen atom, alkyl or phenylalkyl;
R2 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl;
R3 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl or (substituted phenyl)alkyl;
R4 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl;
R5 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl;
R6 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted phenyl)alkyl;
R7 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl;
R8 is an amino acid side chain chosen from the group consisting of natural and unnatural amino acids;
B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, (heteroaryl)alkyl, or a halomethyl group;
a group of the formula:
xe2x80x94CH2XR9;
wherein R9 is phenyl, substituted phenyl, phenylalkyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; and X is an oxygen or a sulfur atom;
a group of the formula:
xe2x80x94CH2xe2x80x94Oxe2x80x94CO-(aryl);
a group of the formula:
xe2x80x94CH2xe2x80x94Oxe2x80x94CO-(heteroaryl);
a group of the formula:
xe2x80x94CH2xe2x80x94Oxe2x80x94POxe2x80x94(R10)R11;
wherein R10 and R11 are independently selected from a group consisting of alkyl, cycloalkyl, phenyl, substituted phenyl, phenylalkyl, and (substituted phenyl)alkyl;
or a pharmaceutically-acceptable salt thereof.
A further aspect of the instant invention are pharmaceutical compositions comprising a compound of the above Formula 1 and a pharmaceutically-acceptable carrier therefor.
Another aspect of this invention involves a method for treating an autoimmune disease comprising administering an effective amount of a pharmaceutical composition discussed above to a patient in need of such treatment.
Yet another aspect of the instant invention is a method of treating an inflammatory disease comprising administering an effective amount of a pharmaceutical composition discussed above to a patient in need of such treatment.
A further aspect of the instant invention is method of treating a neurodegenerative disease comprising administering a pharmaceutically effective amount of a pharmaceutical composition discussed above to a patient in need of such treatment.
Yet another aspect of the instant invention is a method of preventing ischemic injury to a patient suffering from a disease associated with ischemic injury comprising administering an effective amount of a pharmaceutical composition discussed above to a patient in need of such treatment.
One aspect of the instant invention is compounds of the Formula 1: 
wherein:
n is 1 or 2;
m is 1 or 2;
A is R2COxe2x80x94, R3xe2x80x94Oxe2x80x94COxe2x80x94, or R4SO2xe2x80x94;
a group of the formula: 
further wherein:
R1 is a hydrogen atom, alkyl or phenylalkyl;
R2 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl;
R3 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted phenyl)alkyl;
R4 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl) alkyl;
R5 is alkyl, cycloalkyl, (cycloalkyl) alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl) alkyl;
R6 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenylalkyl, or (substituted phenyl)alkyl;
R7 is alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl;
R8 is an amino acid side chain chosen from the group consisting of natural and unnatural amino acids;
B is a hydrogen atom, a deuterium atom, alkyl, cycloalkyl, (cycloalkyl)alkyl, phenyl, phenylalkyl, (substituted)phenyl, (substituted)phenylalkyl, heteroaryl, (heteroaryl)alkyl, or halomethyl;
a group of the formula
xe2x80x94CH2XR9;
wherein R9 is phenyl, phenylalkyl, substituted phenyl, (substituted phenyl)alkyl, heteroaryl, or (heteroaryl)alkyl; and X is an oxygen or a sulfur atom;
a group of the formula:
xe2x80x94CH2xe2x80x94Oxe2x80x94COxe2x80x94(aryl);
a group of the formula:
xe2x80x94CH2xe2x80x94Oxe2x80x94COxe2x80x94(heteroaryl);
a group of the formula:
xe2x80x94CH2xe2x80x94Oxe2x80x94POxe2x80x94(R10)R11;
wherein R10 and R11 are independently selected from a group consisting of alkyl, cycloalkyl, phenyl, substituted phenyl, phenylalkyl, and (substituted phenyl)alkyl;
or a pharmaceutically-acceptable salt thereof.
As used in the above formula, the term xe2x80x9calkylxe2x80x9d means substituted or unsubstituted a straight chain or branched C1 to C8 carbon chain such as methyl, ethyl, tert-butyl, iso-propyl, n-octyl, and the like. Suitable substituents include carboxy, protected carboxy, amino, protected amino, halo, hydroxy, protected hydroxy, nitro, cyano, monosubstituted amino, protected monosubstituted amino, disubstituted amino, C1 to C1 alboxy, C1 to C7 acyl, C1 to C7 acyloxy, and the like.
The term xe2x80x9ccycloalkylxe2x80x9d means a mono-, bi-, or tricyclic saturated ring that is fully saturated or partially unsaturated. Examples of such a group included cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclooctyl, cis- or trans decalin, bicyclo[2.2.1]hept-2-ene, cyclohex-1-enyl, cyclopent-1-enyl, 1,4-cyclooctadienyl, and the like.
The term xe2x80x9c(cycloalkyl)alkylxe2x80x9d means the above-defined alkyl group substituted for one of the above cycloalkyl rings. Examples of such a group include (cyclohexyl)methyl, 3-(cyclopropyl)-n-propyl, 5-(cyclopentyl)hexyl, 6-(adamantyl)hexyl, and the like.
The term xe2x80x9csubstituted phenylxe2x80x9d specifies a phenyl group substituted with one or more, and preferably one or two, moieties chosen from the groups consisting of halogen, hydroxy, protected hydroxy, cyano, nitro, trifluoromethyl, C1 to C7 alkyl, C1 to C7 alkoxy, C1 to C7 acyl, C1 to C7 acyloxy, carboxy, protected carboxy, carboxymethyl, protected carboxymethyl, hydroxymethyl, protected hydroxymethyl, amino, protected amino, (monosubstituted)amino, protected (monosubstituted)amino, (disubstituted)amino, carboxamide, protected carboxamide, N-(C1 to C6 alkyl)carboxamide, protected N-(C1 to C6 alkyl)carboxamide, N,N-di(C1 to C6 alkyl)carboxamide, trifluoromethyl, N-((C1 to C6 alkyl)sulfonyl)amino, N-(phenylsulfonyl)amino or phenyl, substituted or unsubstituted, such that, for example, a biphenyl or naphthyl group results.
Examples of the term xe2x80x9csubstituted phenylxe2x80x9d includes a mono- or di(halo)phenyl group such as 2, 3 or 4-chlorophenyl, 2,6-dichlorophenyl, 2,5-dichlorophenyl, 3,4-dichlorophenyl, 2, 3 or 4-bromophenyl, 3,4-dibromophenyl, 3-chloro-4-fluorophenyl, 2, 3 or 4-fluorophenyl and the like; a mono or di(hydroxy)phenyl group such as 2, 3, or 4-hydroxyphenyl, 2,4-dihydroxyphenyl, the protected-hydroxy derivatives thereof and the like; a nitrophenyl group such as 2, 3, or 4-nitrophenyl; a cyanophenyl group, for example, 2, 3 or 4-cyanophenyl; a mono- or di(alkyl)phenyl group such as 2, 3, or 4-methylphenyl, 2,4-dimethylphenyl, 2, 3 or 4-(iso-propyl)phenyl, 2, 3, or 4-ethylphenyl, 2, 3 or 4-(n-propyl)phenyl and the like; a mono or di(alkoxy)phenyl group, for example, 2,6-dimethoxyphenyl, 2, 3 or 4-(isopropoxy)phenyl, 2, 3 or 4-(t-butoxy)phenyl, 3-ethoxy-4-methoxyphenyl and the like; 2, 3 or 4-trifluoromethylphenyl; a mono- or dicarboxyphenyl or (protected carboxy)phenyl group such as 2, 3 or 4-carboxyphenyl or 2,4-di(protected carboxy)phenyl; a mono- or di(hydroxymethyl)phenyl or (protected hydroxymethyl)phenyl such as 2, 3 or 4-(protected hydroxymethyl)phenyl or 3,4-di(hydroxymethyl)phenyl; a mono- or di(aminomethyl)phenyl or (protected aminomethyl)phenyl such as 2, 3 or 4-(aminomethyl)phenyl or 2,4-(protected aminomethyl)phenyl; or a mono- or di(N-(methylsulfonylamino))phenyl such as 2, 3 or 4-(N-(methylsulfonylamino))phenyl. Also, the term xe2x80x9csubstituted phenylxe2x80x9d represents disubstituted phenyl groups wherein the substituents are different, for example, 3-methyl-4-hydroxyphenyl, 3-chloro-4-hydroxyphenyl, 2-methoxy-4-bromophenyl, 4-ethyl-2-hydroxyphenyl, 3-hydroxy-4-nitrophenyl, 2-hydroxy-4-chlorophenyl and the like.
The term xe2x80x9c(substituted phenyl)alkylxe2x80x9d means one of the above substituted phenyl groups attached to one of the above-described alkyl groups. Examples of include such groups as 2-phenyl-1-chloroethyl, 2-(4xe2x80x2-methoxyphenyl)ethyl, 4-(2xe2x80x2,6xe2x80x2-dihydroxy phenyl)n-hexyl, 2-(5xe2x80x2-cyano-3xe2x80x2-methoxyphenyl)n-pentyl, 3-(2xe2x80x2,6xe2x80x2-dimethylphenyl)n-propyl, 4-chloro-3-aminobenzyl, 6-(4xe2x80x2-methoxyphenyl)-3-carboxy(n-hexyl), 5-(4xe2x80x2-aminomethylphenyl)-3-(aminomethyl)n-pentyl, 5-phenyl-3-oxo-n-pent-1-yl, (4-hydroxynapth-2-yl)methyl and the like.
The terms xe2x80x9chaloxe2x80x9d and xe2x80x9chalogenxe2x80x9d refer to the fluoro, chloro, bromo or iodo groups. There can be one or more halogen, which are the same or different. Preferred halogens are chloro and fluoro.
The term xe2x80x9carylxe2x80x9d refers to aromatic five and six membered carbocyclic rings. Six membered rings are preferred.
The term xe2x80x9cheteroarylxe2x80x9d denotes optionally substituted five-membered or six-membered rings that have 1 to 4 heteroatoms, such as oxygen, sulfur and/or nitrogen atoms, in particular nitrogen, either alone or in conjunction with sulfur or oxygen ring atoms. These five-membered or six-membered rings are fully unsaturated.
Furthermore, the above optionally substituted five-membered or six-membered rings can optionally be fused to a aromatic 5-membered or 6-membered ring system. For example, the rings can be optionally fused to an aromatic 5-membered or 6-membered ring system such as a pyridine or a triazole system, and preferably to a benzene ring.
The following ring systems are examples of the heterocyclic (whether substituted or unsubstituted) radicals denoted by the term xe2x80x9cheteroarylxe2x80x9d: thienyl, furyl, pyrrolyl, pyrrolidinyl, imidazolyl, isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl, thiatriazolyl, oxatriazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, oxazinyl, triazinyl, thiadiazinyl tetrazolo, 1,5-[b]pyridazinyl and purinyl, as well as benzo-fused derivatives, for example, benzoxazolyl, benzthiazolyl, benzimidazolyl and indolyl.
Substituents for the above optionally substituted heteroaryl rings are from one to three halo, trihalomethyl, amino, protected amino, amino salts, mono-substituted amino, di-substituted amino, carboxy, protected carboxy, carboxylate salts, hydroxy, protected hydroxy, salts of a hydroxy group, lower alkoxy, lower alkylthio, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, (cycloalkyl)alkyl, substituted (cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, and (substituted phenyl)alkyl. Substituents for the heteroaryl group are as heretofore defined, or in the case of trihalomethyl, can be trifluoromethyl, trichloromethyl, tribromomethyl, or triiodomethyl. As used in conjunction with the above substituents for heteroaryl rings, xe2x80x9clower alkoxyxe2x80x9d means a C1 to C4 alkoxy group, similarly, xe2x80x9clower alkylthioxe2x80x9d means a C1 to C4 alkylthio group. The term xe2x80x9csubstituted alkylxe2x80x9d means the above defined alkyl group substituted from one to three times by a hydroxy, protected hydroxy, amino, protected amino, cyano, halo, trifloromethyl, mono-substituted amino, di-substituted amino, lower alkoxy, lower alkylthio, carboxy, protected carboxy, or a carboxy, amino, and/or hydroxy salt. As used in conjunction with the substituents for the heteroaryl rings, the terms xe2x80x9csubstituted (cycloalkyl)alkylxe2x80x9d and xe2x80x9csubstituted cycloalkylxe2x80x9d are as defined above substituted with the same groups as listed for a xe2x80x9csubstituted alkylxe2x80x9d group. The term xe2x80x9c(monosubstituted)aminoxe2x80x9d refers to an amino group with one substituent chosen from the group consisting of phenyl, substituted phenyl, alkyl, substituted alkyl, C1 to C7 acyl, C2 to C7 alkenyl, C2 to C7 substituted alkenyl, C2 to C7 alkynyl, C7 to C16 alkylaryl, C7 to C16 substituted alkylaryl and heteroaryl group. The (monosubstituted) amino can additionally have an amino-protecting group as encompassed by the term xe2x80x9cprotected (monosubstituted)amino.xe2x80x9d The term xe2x80x9c(disubstituted)aminoxe2x80x9d refers to amino groups with two substituents chosen from the group consisting of phenyl, substituted phenyl, alkyl, substituted alkyl, C1 to C7 acyl, C2 to C7 alkenyl, C2 to C7 alkynyl, C7 to C16 alkylaryl, C7 to C16 substituted alkylaryl and heteroaryl. The two substituents can be the same or different. The term xe2x80x9cheteroaryl(alkyl)xe2x80x9d denotes an alkyl group as defined above, substituted at any position by a heteroaryl group, as above defined.
The term xe2x80x9cpharmaceutically-acceptable saltxe2x80x9d encompasses those salts that form with the carboxylate anions and includes salts formed with the organic and inorganic cations such as those chosen from the alkali and alkaline earth metals, (for example, lithium, sodium, potassium, magnesium, barium and calcium); ammonium; and the organic cations (for example, dibenzylammonium, benzylammonium, 2-hydroxyethylammonium, bis(2-hydroxyethyl)ammonium, phenylethylbenzylammonium, dibenzylethylenediammonium, and like cations.) Other cations encompassed by the above term include the protonated form of procaine, quinine and N-methylglucosamine, the protonated forms of basic amino acids such as glycine, ornithine, histidine, phenylglycine, lysine, and arginine, and acetic acid-like counter-ions such as acetate and trifluoroacetate. Furthermore, any zwitterionic form of the instant compounds formed by a carboxylic acid and an amino group is referred to by this term. A preferred cation for the carboxylate anion is the sodium cation. Furthermore, the term includes salts that form by standard acid-base reactions with basic groups (such as amino groups) and includes organic or inorganic acids. Such acids include hydrochloric, sulfuric, phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, palmitic, cholic, pamoic, mucic, D-glutamic, D-camphoric, glutaric, phthalic, tartaric, lauric, stearic, salicyclic, methanesulfonic, benzenesulfonic, sorbic, picric, benzoic, cinnamic, and the like acids.
The compounds of Formula 1 may also exist as solvates and hydrates. Thus, these compounds may crystallize with, for example, waters of hydration, or one, a number of, or any fraction thereof of molecules of the mother liquor solvent. The solvates and hydrates of such compounds are included within the scope of this invention.
The term xe2x80x9ccarboxy-protecting groupxe2x80x9d as used herein refers to one of the ester derivatives of the carboxylic acid group commonly employed to block or protect the carboxylic acid group while reactions are carried out on other functional groups on the compound. Examples of such carboxylic acid protecting groups include t-butyl, 4-nitrobenzyl, 4-methoxybenzyl, 3,4-dimethoxybenzyl, 2,4-dimethoxybenzyl, 2,4,6-trimethoxybenzyl, 2,4,6-trimethylbenzyl, pentamethylbenzyl, 3,4-methylenedioxybenzyl, benzhydryl, 4,4xe2x80x2-dimethoxytrityl, 4,4xe2x80x2,4xe2x80x3-trimethoxytrityl, 2-phenylpropyl, trimethylsilyl, t-butyldimethylsilyl, phenacyl, 2,2,2-trichloroethyl, xcex2-(trimethylsilyl)ethyl, xcex2-(di(n-butyl)methylsilyl)ethyl, p-toluenesulfonylethyl, 4-nitrobenzylsulfonylethyl, allyl, cinnamyl, 1-(trimethylsilylmethyl)-propenyl and like moieties. The species of carboxy-protecting group employed is not critical so long as the derivatized carboxylic acid is stable to the conditions of subsequent reaction(s) and can be removed at the appropriate point without disrupting the remainder of the molecule. Further examples of these groups are found in C. B. Reese and E. Haslam, xe2x80x9cProtective Groups in Organic Chemistry,xe2x80x9d J. G. W. McOmie, Ed., Plenum Press, New York, N.Y., 1973, Chapter 5, respectively, and T. W. Greene and P. G. M. Wuts, xe2x80x9cProtective Groups in Organic Synthesis,xe2x80x9d 2nd ed., John Wiley and Sons, New York, N.Y., 1991, Chapter 5, each of which is incorporated herein by reference. A related term is xe2x80x9cprotected carboxy,xe2x80x9d which refers to a carboxy group substituted with one of the above carboxy-protecting groups.
The term xe2x80x9chydroxy-protecting groupxe2x80x9d refers to readily cleavable groups bonded to hydroxyl groups, such as the tetrahydropyranyl, 2-methoxyprop-2-yl, 1-ethoxyeth-1-yl, methoxymethyl, xcex2-methoxyethoxymethyl, methylthiomethyl, t-butyl, t-amyl, trityl, 4-methoxytrityl, 4,4xe2x80x2-dimethoxytrityl, 4,4xe2x80x2,4xe2x80x3-trimethoxytrityl, benzyl, allyl, trimethylsilyl, (t-butyl)dimethylsilyl, 2,2,2-trichloroethoxycarbonyl groups and the like.
Further examples of hydroxy-protecting groups are described by C. B. Reese and E. Haslam, xe2x80x9cProtective Groups in Organic Chemistry,xe2x80x9d J. G. W. McOmie, Ed., Plenum Press, New York, N.Y., 1973, Chapters 3 and 4, respectively, and T. W. Greene and P. G. M. Wuts, xe2x80x9cProtective Groups in Organic Synthesis,xe2x80x9d Second Edition, John Wiley and Sons, New York, N.Y., 1991, Chapters 2 and 3. A preferred hydroxy-protecting group is the tert-butyl group. The related term xe2x80x9cprotected hydroxyxe2x80x9d denotes a hydroxy group bonded to one of the above hydroxy protecting groups.
The term xe2x80x9camino-protecting groupxe2x80x9d as used herein refers to substituents of the amino group commonly employed to block or protect the amino functionality while reacting other functional groups of the molecule. The term xe2x80x9cprotected (monosubstituted)aminoxe2x80x9d means there is an amino-protecting group on the monosubstituted amino nitrogen atom.
Examples of such amino-protecting groups include the formyl (xe2x80x9cForxe2x80x9d) group, the trityl group, the phthalimido group, the trichloroacetyl group, the trifluoroacetyl group, the chloroacetyl, bromoacetyl, and iodoacetyl groups, urethane-type blocking groups, such as t-butoxycarbonyl (xe2x80x9cBocxe2x80x9d), 2-(4-biphenylyl)propyl-2-oxycarbonyl (xe2x80x9cBpocxe2x80x9d), 2-phenylpropyl-2-oxycarbonyl (xe2x80x9cPocxe2x80x9d), 2-(4-xenyl)isopropoxycarbonyl, 1,1-diphenylethyl-1-oxycarbonyl, 1,1-diphenylpropyl-1-oxycarbonyl, 2-(3,5-dimethoxyphenyl)propyl-2-oxycarbonyl (xe2x80x9cDdzxe2x80x9d), 2-(p-toluyl)propyl-2-oxycarbonyl, cyclopentanyloxycarbonyl, 1-methylcyclopentanyloxycarbonyl, cyclohexanyloxycarbonyl, 1-methylcyclohexanyloxycarbonyl, 2-methylcyclohexanyloxycarbonyl, 2-(4-toluylsulfonyl)ethoxycarbonyl, 2-(methylsulfonyl)ethoxycarbonyl, 2-(triphenylphosphino)ethoxycarbonyl, 9-fluorenylmethoxycarbonyl (xe2x80x9cFmocxe2x80x9d), 2-(trimethylsilyl)ethoxycarbonyl, allyloxycarbonyl, 1-(trimethylsilylmethyl)prop-1-enyloxycarbonyl, 5-benzisoxalylmethoxycarbonyl, 4-acetoxybenzyloxycarbonyl, 2,2,2-trichloroethoxycarbonyl, 2-ethynyl-2-propoxycarbonyl, cyclopropylmethoxycarbonyl, isobornyloxycarbonyl, 1-piperidyloxycarbonyl, benzyloxycarbonyl (xe2x80x9cCbzxe2x80x9d), 4-phenylbenzyloxycarbonyl, 2-methylbenzyloxy-carbonyl, xcex1-2,4,5,-tetramethylbenzyloxycarbonyl (xe2x80x9cTmzxe2x80x9d), 4-methoxybenzyloxycarbonyl, 4-fluorobenzyloxycarbonyl, 4-chlorobenzyloxycarbonyl, 3-chlorobenzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl, 3-bromobenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl, 4-cyanobenzyloxycarbonyl, 4-(decyloxy)benzyloxycarbonyl and the like; the benzoylmethylsulfonyl group, 2,2,5,7,8-pentamethylchroman-6-sulfonyl group (xe2x80x9cPMCxe2x80x9d) dithiasuccinoyl (xe2x80x9cDtsxe2x80x9d), the 2-(nitro)phenylsulfenyl group (xe2x80x9cNpsxe2x80x9d), the diphenyl-phosphine oxide group and like amino-protecting groups. The species of amino-protecting group employed is not critical so long as the derivatized amino group is stable to the conditions of the subsequent reaction(s) and can be removed at the appropriate point without disrupting the remainder of the molecule. Preferred amino-protecting groups are Boc, Cbz and Fmoc. Further examples of amino-protecting groups embraced by the above term are well known in organic synthesis and the peptide art and are described by, for example, T. W. Greene and P. G. M. Wuts, xe2x80x9cProtective Groups in Organic Synthesis,xe2x80x9d 2nd ed., John Wiley and Sons, New York, N.Y., 1991, Chapter 7, M. Bodanzsky, xe2x80x9cPrinciples of Peptide Synthesis,xe2x80x9d 1st and 2nd revised ed., Springer-Verlag, New York, N.Y., 1984 and 1993, and J. M. Stewart and J. D. Young, xe2x80x9cSolid Phase Peptide Synthesis,xe2x80x9d 2nd ed., Pierce Chemical Co., Rockford, Ill., 1984, E. Atherton and R. C. Shephard, xe2x80x9cSolid Phase Peptide Synthesisxe2x80x94A Practical Approachxe2x80x9d IRL Press, Oxford, England (1989), each of which is incorporated herein by reference. The related term xe2x80x9cprotected aminoxe2x80x9d defines an amino group substituted with an amino-protecting group discussed above.
The terms xe2x80x9cnatural and unnatural amino acidsxe2x80x9d (xcex1-amino acid) refers to both the naturally occurring amino acids and other xe2x80x9cnon-proteinxe2x80x9d xcex1-amino acids commonly utilized by those in the peptide chemistry arts when preparing synthetic analogues of naturally occurring peptides, including D and L forms. The naturally occurring amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, xcex3-carboxyglutamic acid, arginine, ornithine and lysine. Examples of xe2x80x9cnon-proteinxe2x80x9d alpha-amino acids include hydroxylysine, citrulline, kynurenine, (4-aminophenyl)alanine, 3-(2xe2x80x2-naphthyl)alanine, 3-(1xe2x80x2-naphthyl)alanine, methionine sulfone, (t-butyl)alanine, (t-butyl)glycine, 4-hydroxyphenylglycine, aminoalanine, phenylglycine, vinylalanine, propargyl-gylcine, 1,2,4-triazolo-3-alanine, thyronine, 6-hydroxytryptophan, 5-hydroxytryptophan, 3-hydroxykynurenine, 3-aminotyrosine, trifluoromethyl-alanine, 2-thienylalanine, (2-(4-pyridyl)ethyl)cysteine, 3,4-dimethoxy-phenylalanine, 3-(2xe2x80x2-thiazolyl)alanine, ibotenic acid, 1-amino-1-cyclopentane-carboxylic acid, 1-amino-1-cyclohexanecarboxylic acid, quisqualic acid, 3-(trifuoromethylphenyl)alanine, (cyclohexyl)glycine, thiohistidine, 3-methoxytyrosine, elastatinal, norleucine, norvaline, alloisoleucine, homoarginine, thioproline, dehydroproline, hydroxyproline, homoproline, xcex1-amino-n-butyric acid, cyclohexylalanine, 2-amino-3-phenylbutyric acid, phenylalanine substituted at the ortho, meta, or para position of the phenyl moiety with one or two of the following: a (C1 to C4) alkyl, a (C1 to C4)alkoxy, halogen or nitro groups or substituted with a methylenedioxy group; xcex2-2- and 3-thienylalanine, xcex2-2- and 3-furanylalanine, xcex2-2-, 3- and 4-pyridylalanine, xcex2-(benzothienyl-2- and 3-yl)alanine, xcex2-(1- and 2-naphthyl)alanine, O-alkylated derivatives of serine, threonine or tyrosine, S-alkylated cysteine, S-alkylated homocysteine, O-sulfate, O-phosphate and O-carboxylate esters of tyrosine, 3-(sulfo)tyrosine, 3-(carboxy)tyrosine, 3-(phospho)tyrosine, the 4-methane sulfonic acid ester of tyrosine, 4-methane phosphonic acid ester of tyrosine, 3,5-diiodotyrosine, 3-nitrotyrosine, xcex5-alkyl lysine, and delta-alkyl ornithine. Any of these xcex1-amino acids may be substituted with a methyl group at the alpha position, a halogen at any aromatic residue on the xcex1-amino side chain, or an appropriate protective group at the O, N, or S atoms of the side chain residues. Appropriate protective groups are discussed above.
Depending on the choice of solvent and other conditions known to the practitioner skilled in the art, compounds of this invention may also take the hemi-ketal, hemi-acetal, ketal or acetal form, which forms are included in the instant invention.
In addition, it should be understood that the equilibrium forms of the compounds of this invention may include tautomeric forms. All such forms of these compounds are expressly included in the present invention.
Also, it will be understood by those skilled in the art that when B in Formula 1 is a hydrogen atom, a semicarbazone may be formed with the resulting aldehyde. Such semicarbazones are also included as compounds of Formula 1, as well as the pharmaceutical compositions containing those compounds. Such semicarbazones include, for example, semicarbazone derivative of the optimal groups and embodiments of the 4-oxo butanoic acid derivatives of the oxoazepino indole and oxo-azepino quinoline compounds set forth below.
An optimal group of compounds of Formula 1 exists when in the above Formula n is 1, and more so when m is 1. These compounds will be referred to herein as the xe2x80x9coxoazepino indolexe2x80x9d compounds.
An optimal group of oxoazepino indole compounds exists when B in Formula 1 is a hydrogen atom, compounds referred to herein as xe2x80x9c4-oxobutanoic derivativesxe2x80x9d. One embodiment of note of such 4-oxobutanoic acid derivatives occurs when A in Formula 1 is a group of the formula R2COxe2x80x94, more so when R2 is 2-(carboxy)eth-1-yl, 2-(phenyl)eth-1-yl, methyl, napth-1-yl or phenyl, and especially so when R1 is a hydrogen atom. Another embodiment of 4-oxobutanoic acid derivatives exists when A is a group of the formula R5xe2x80x94COxe2x80x94NHxe2x80x94CHR8xe2x80x94COxe2x80x94, more so when R5 is a methyl group and R8 is a group of the formula xe2x80x94CH2COOH, and especially so when R1 is a hydrogen atom. Yet another embodiment of 4-oxobutanoic acid derivatives of note has A as a group of the formula R6xe2x80x94Oxe2x80x94COxe2x80x94NHxe2x80x94CHR8xe2x80x94COxe2x80x94, further wherein R6 is a benzyl group and R8 is a group of the formula xe2x80x94CH2xe2x80x94COOH, and especially so when R1 is a hydrogen atom. Still another embodiment of the 4-oxobutanoic derivatives occurs when A is a group of the formula R3xe2x80x94Oxe2x80x94COxe2x80x94 wherein R3 is benzyl, and especially so when R1 is a hydrogen atom.
Another optimal group of oxoazepino indole compounds occurs when B in Formula 1 is a monofluoromethyl group and are thus referred to as xe2x80x9cmonofluoromethyl derivatives.xe2x80x9d A noteworthy embodiment of monofluoromethyl derivatives has A as a group of the formula R3xe2x80x94Oxe2x80x94COxe2x80x94, more so when R3 is a benzyl group, and especially so when R1 is a hydrogen atom.
A further optimal group of oxoazepino indole compounds occurs when B in Formula 1 is a group of the formula xe2x80x94CH2xe2x80x94Oxe2x80x94PO(R10)R11. This group of compounds is referred to herein as xe2x80x9cphosphinyloxy derivativesxe2x80x9d. A group of noteworthy phosphinyloxy derivatives has R10 and R11 each as phenyl groups. An embodiment of such diphenyl phosphinyloxy compounds exists wherein A in Formula 1 is a group of the formula
R3xe2x80x94Oxe2x80x94COxe2x80x94 wherein R3 is benzyl, and especially so when R1 is a hydrogen atom.
Another optimal group of compounds of Formula 1 exists when in the above Formula n is 2, and more so when m is 1. These compounds will be referred to herein as the xe2x80x9coxoazepino quinolinexe2x80x9d compounds.
An exemplary group of oxoazepino quinoline compounds occurs when B in Formula 1 is a hydrogen atom, more so when A is a group of the formula R3xe2x80x94Oxe2x80x94COxe2x80x94 wherein R3 is benzyl, and especially so when R1 is a hydrogen atom.
It should be understood that the compounds of this invention may be modified by appropriate functionalities to enhance selective biological properties. Such modifications are known in the art and include those which increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter rate of exertion. In addition, the compounds may be altered to pro-drug form such that the desired compound is created in the body of the patient as the result of the action of metabolic or other biochemical processes on the pro-drug. Some examples of pro-drug forms include ketal, acetal, oxime, and hydrazone forms of compounds which contain ketone or aldehyde groups, especially where they occur in the A group or the modified aspartic or glutamic residues attached to the tricyclic nucleus of the compounds of this invention.
The compounds of Formula 1 of this invention may be synthesized using conventional techniques. Advantageously, these compounds are conveniently synthesized from readily available starting materials.
Thus, compounds of the instant invention can be synthesized in general by combining a tricyclic nucleus set forth below in Formula 2: 
with the modified aspartic and glutamic acid residues of Formula 3a-d: 
in the presence of a standard peptide coupling agents such as dicyclohexylcarbodiimide (DCC)/1-hydroxybenzotriazole (HOBt), BOP reagent, pyBOP, TBTU, EEDQ, 1-ethyl(3,3xe2x80x2-dimethyl-1xe2x80x2-aminopropyl)carbodiimide (EDAC)/HOBt, and the like, as discussed in J. Jones, xe2x80x9cAmino Acid and Peptide Synthesis,xe2x80x9d Steven G. Davis ed., Oxford University Press, Oxford, pp. 25-41 (1992), herein incorporated by reference. In the above formula, APG is an amino protecting group. The amino protecting group is then removed and the resulting amine is combined with the substituted acyl group of Formula 4:
Rcxe2x80x94COxe2x80x94Xxe2x80x83xe2x80x83FORMULA 4
or the sulfonyl group of Formula 5:
R4SO2xe2x80x94X.xe2x80x83xe2x80x83FORMULA 5
In the above formulas, R1 is as defined above, and Rc is R2, R3xe2x80x94O, R4, or any of the side chains containing R8 as defined for group A in Formula 1. Of course, such moieties would have any hydroxy, carboxy or amino groups in the protected form so as not to interfere with the coupling reaction (Formula 3a-d), the acylation reaction (Formula 4) or the sulfonation reaction (Formula 5). X in the above Formulas represents a facile leaving group for the acylation or sulfonation reactions.
In the case where the coupling reaction was carried out with the amino alcohol of Formula 3c, the alcohol moiety must be oxidized to the corresponding carbonyl compound prior to removal of the protecting groups. Preferred methods for the oxidation reaction include Swern oxidation (oxalyl chloride-dimethyl sulfoxide, methylene chloride at xe2x88x9278xc2x0 C. followed by triethylmine; and Dess-Martin oxidation (Dess-Martin periodinane, t-butanol, and methylene chloride.) The protecting groups contained in substructures of the Formula 2 and 3a-d are removed by methods well known in the art.
The tricyclic nucleus of Formula 2 is synthesized by methods known in the art. For example, see D. S. Karanewsky, U.S. Pat. No. 5,504,080 issued Apr. 2, 1996; J. A. Robl et al., Tetrahedron Letters, 36:1593-1596 (1995); and S. De Lombaert et al., Tetrahedron Letters, 35:7513-7516 (1994), all of which are incorporated herein by reference.
The modified aspartic or glutamic acid for Formula 3a-d can be elaborated by methods well known in the art. See, for example, European Patent Application 519,748; PCT Patent Application No. PCT/EP92/02472; PCT Patent Application No. PCT/US91/06595; PCT Patent Application No. PCT/US391/02339; European Patent Application No. 623,592; World Patent Application No. WO 93/09135; PCT Patent Application No. PCT/US94/08868; European Patent Application No. 623,606; European Patent Application No. 618,223; European Patent Application No. 533,226; European Patent Application No. 528,487; European Patent Application No. 618,233; PCT Patent Application No. PCT/EP92/02472, World Patent Application No. WO 93/09135; PCT Patent Application No. PCT/US93/03589; and PCT Patent Application No. PCT/US93/00481, all of which are herein incorporated by reference.
The acyl group of Formula 4 and the corresponding R4SO2 groups are also synthesized by methods well known in the art. See, for example, U.S. Pat. No. 5,504,080, issued Apr. 2, 1996, herein incorporated by reference. While this group can be elaborated once bonded to the tricyclic nucleus, it is preferable that it be intact before being attached to the nucleus.
Once the side chains of Formula 3 and Formula 4 or Formula 5 are bonded to the tricyclic nucleus of Formula 2, one skilled in the art would usually remove any amino, hydroxy, or carboxy-protecting groups to enhance the activity of the synthesized molecule.
Pharmaceutical compositions of this invention comprise any of the compounds of Formula 1, and pharmaceutically acceptable salts thereof, with any pharmaceutically acceptable carrier, adjuvant or vehicle (hereinafter collectively referred to as xe2x80x9cpharmaceutically-accetable carriersxe2x80x9d). Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchange, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as the various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyarylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
An optimal group of pharmaceutical compositions of Formula 1 exists when in the above Formula n is 1, and more so when m is 1. These compositions will be referred to herein as the xe2x80x9coxoazepino indolexe2x80x9d compostions.
An optimal group of oxoazepino indole compositions exists when B in Formula 1 is a hydrogen atom, compositions referred to herein as xe2x80x9c4-oxobutanoic derivativesxe2x80x9d. One embodiment of note of such 4-oxobutanoic acid derivatives occurs when A in Formula 1 is a group of the formula R2COxe2x80x94, more so when R2 is 2-(carboxy)eth-1-yl, 2-(phenyl)eth-1-yl, methyl, napth-1-yl or phenyl, and especially so when R1 is a hydrogen atom.
Another embodiment of 4-oxobutanoic acid derivatives exists when A is a group of the formula R5xe2x80x94COxe2x80x94NHxe2x80x94CHR8xe2x80x94COxe2x80x94, more so when R5 is a methyl group and R8 is a group of the formula xe2x80x94CH2COOH, and especially so when R1 is a hydrogen atom. Yet another embodiment of 4-oxobutanoic acid derivatives of note has A as a group of the formula R6xe2x80x94Oxe2x80x94COxe2x80x94NHxe2x80x94CHR8xe2x80x94COxe2x80x94, further wherein R6 is a benzyl group and R8 is a group of the formula xe2x80x94CH2xe2x80x94COOH, and especially so when R1 is a hydrogen atom. Still another embodiment of the 4-oxobutanoic derivatives occurs when A is a group of the formula R3xe2x80x94Oxe2x80x94COxe2x80x94 wherein R3 is benzyl, and especially so when R1 is a hydrogen atom.
Another optimal group of oxoazepino indole compositions occurs when B in Formula 1 is a monofluoromethyl group and are thus referred to as xe2x80x9cmonofluoromethyl derivatives.xe2x80x9d A noteworthy embodiment of monofluoromethyl derivatives has A as a group of the formula R3xe2x80x94Oxe2x80x94COxe2x80x94, more so when R3 is a benzyl group, and especially so when R1 is a hydrogen atom.
A further optimal group of oxoazepino indole compositions occurs when B in Formula 1 is a group of the formula xe2x80x94CH2xe2x80x94Oxe2x80x94PO(R10)R11. This group of compositions is referred to herein as xe2x80x9cphosphinyloxy derivativesxe2x80x9d. A group of noteworthy phosphinyloxy derivatives has R10 and R11 each as phenyl groups. An embodiment of such diphenyl phosphinyloxy compositions exists wherein A in Formula 1 is a group of the formula R3xe2x80x94Oxe2x80x94COxe2x80x94 wherein R3 is benzyl, and especially so when R1 is a hydrogen atom.
Another optimal group of pharmaceutical compositions of Formula 1 exists when in the above Formula n is 2, and more so when m is 1. These compositions will be referred to herein as the xe2x80x9coxoazepino quinolinexe2x80x9d compositions.
An exemplary group of oxoazepino quinoline compositions occurs when B in Formula 1 is a hydrogen atom, more so when A is a group of the formula R3xe2x80x94Oxe2x80x94COxe2x80x94 wherein R3 is benzyl, and especially so when R1 is a hydrogen atom.
The pharmaceutical compositions of this invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implated reservoir. Oral and parenteral administration are preferred. The term xe2x80x9cparenteralxe2x80x9d as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer""s solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant such as Ph. Helv or a similar alcohol.
The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspensions and solutions. In the case of tablets for oral use, carrier which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in capsule form useful diluents include lactose and dried corn starch. When aqueous suspension are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.
The pharmaceutical compositions of this invention may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.
Topical administration of the pharmaceutical compositions of this invention is especially useful when the desired treatment involves areas or organs readily accessible to topical application. For application topically to the skin, the pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of this invention may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topically-transdermal patches are also included in this invention.
The pharmaceutical compositions of this invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
The pharmaceutical compositions of this invention may be employed in a conventional manner for the treatment of diseases which are mediated by IL-1 in mammals. Such methods of treatment, their dosage levels and requirements may be selected by those of ordinary skill in the art form available methods and techniques. For example, a pharmaceutical composition of this invention may be administered to a patient suffering from an IL-1 mediated disease in a pharmaceutically acceptable manner and in an amount effective to lessen the severity of that disease.
In addition, the compounds of this invention may be used in combination with either conventional anti-inflammatory agents or with matrix metalloprotease inhibitors, lipoxygenase inhibitors and antagonists of cytokines other than IL-1xcex2.
The compounds of this invention can also be administered in combination with immunomodulators (e.g., bropirimine, anti-human alpha interferon antibody, IL-2, GM-CSF, methionine enkephalin, interferon alpha, diethyldithiocarbamate, tumor necrosis factor, naltrexons and rEPO) or with prostaglandins, to prevent or combat IL-1-mediated disease symptoms such as inflammation.
When the compounds of this invention are administered in combination therapies with other agents, they may be administered sequentially or concurrently to the patient. Alternatively, pharmaceutical compositions according to this invention may be comprised of a combination of a compound of Formula 1 and another therapeutic or prophylactic agent.
The disease states which may be treated or prevented by the instant pharmaceutical compositions include, but are not limited to, inflammatory diseases, autoimmune diseases and neurodegenerative diseases, and for inhibiting unwanted apoptosis involved in ischemic injury, such as ischemic injury to the heart (e.g., myocardial infarction), brain (e.g., stroke), and kidney (e.g., ischemic kidney disease). Methods of administering an effective amount of the above-described pharmaceutical compositions to mammals, also referred to herein as patients, in need of such treatment (that is, those suffering from inflammatory diseases, autoimmune diseases, and neurodegenerative diseases,) are additional aspects of the instant invention.
Yet another aspect of the instant invention is a method of preventing ischemic injury to a patient suffering from a disease associated with ischemic injury comprising administering an effective amount of a pharmaceutical composition discussed above to a patient in need of such treatment.
Also, each of the methods directed to methods for treating inflammatory diseases, autoimmune diseases, neurodegenerative disease, and preventing ischemic injury emcompass using any of the optimal groups and embodiments of pharmaceutical compositions set forth above.
Inflammatory disease which may be treated or prevented include, for example, septic shock, septicemia, and adult respiratory distress syndrome. Target autoimmune diseases include, for example, rheumatoid, arthritis, systemic lupus erythematosus, scleroderma, chronic thyroiditis, Graves"" disease, autoimmune gastritis, insulin-dependent diabetes mellitus, autoimmune hemolytic anemia, autoimmune neutropenia, thrombocytopenia, chronic active hepatitis, myasthenia gravis and multiple sclerosis. Target neurodegenerative diseases include, for example, amyotrophic lateral sclerosis, Alzheimer""s disease, Parkinson""s disease, and primary lateral sclerosis. The pharmaceutical compositions of this invention may also be used to promote wound healing. Target diseases associated with harmful, thus unwanted apoptosis, in other words, those associated with ischemic injury, includes myocardial infarction, stroke, and ischemic kidney disease. The pharmaceutical compositions of this invention may also be used to treat infectious diseases, especially those involved with viral infections.
The term xe2x80x9ceffective amountxe2x80x9d refers to dosage levels of the order of from about 0.05 mg to about 140 mg per kilogram of body weight per day for use in the treatment of the above-indicated conditions (about 2.5 mg to about 7 gms. per patient per day). For example, inflammation may be effectively treated by the administration of from about 0.01 to 50 mg of the compound per kilogram of body weight per day (about 0.5 mg to about 3.5 gms per patient per day).
The amount of the compounds of Formula 1 that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a formulation intended for the oral administration of humans may contain from 0.5 mg to 5 gm of a compound of Formula 1 compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to about 95 percent of the total composition. Dosage unit forms will generally contain between from about 1 mg to about 500 mg of an active compound of Formula 1.
It will be understood, however, that the specific xe2x80x9ceffective amountxe2x80x9d for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
Although this invention focuses on the use of the compounds disclosed herein for preventing and treating IL-1-mediated diseases, the compounds of this invention can also be used as inhibitory agents for other cysteine proteases.
The compounds of this invention are also useful as commercial reagents which effectively bind to the ICE/ced-3 family of cysteine protease or other cysteine proteases. As commercial reagents, the compounds of this invention, and their derivatives, may be used to block proteolysis of a target peptide or may be derivatized to bind to a stable resin as a tethered substrate for affinity chromatography applications. These and other uses which characterize commercial cystine protease inhibitors will be evident to those of ordinary skill in the art.
In order that this invention be more fully understood, the following examples are set forth. These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any way.
In the following Examples, proton NMR spectra were obtained at 300 MHZ; chemical shifts are quoted downfield from internal tetramethylsilane.

Preparation of (2S-cis)-5-Benzyloxycarbonylamino-1,2,4,5,6,7-Hexahydro-4-Oxoazepino[3,2,1-hi]indole-2-Carboxylic Acid, Ethyl Ester
To a solution of (2S-cis)-5-amino-1,2,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carboxylic acid, ethyl ester (0.437 g, 1.73 mmol, prepared as described in Tetrahedron Letters 36, pp. 1593-1596 (1995) and U.S. Pat. No. 5,504,080 (Apr. 2, 1996)) in methylene chloride (4 mL) stirring at 0xc2x0 C. was added benzyl chloroformate (0.370 mL, 2.6 mmol) and triethylamine (0.724 mL, 5.2 mmol) and the resulting mixture was stirred under nitrogen for 45 minutes. The reaction was quenched with water then partitioned between ethyl acetate and 5% aqueous potassium bisulfate solution. The aqueous layer was back-extracted two times with ethyl acetate, then the combined organic layers were washed with saturated sodium chloride solution, dried over sodium sulfate and evaporated to dryness. Purification of the crude product by flash chromatography on silica gel (S/P brand silica gel 60 xc3x85, 230-400 mesh ASTM) eluting with ethyl acetate-hexane (2:1) gave 0.558 g (68%) of crude product. Trituration with ethyl acetate-hexane (1:4) gave 0.480 g of the title compound as white solid; m.p.: 139-140xc2x0 C. TLC (ethyl acetate-hexane, 2:1): Rf=0.6; 1H-NMR (300 MHz, CDCl3): xcex4 7.35-7.30 (m, 5H), 7.02-6.94 (m, 3H), 6.17 (d, J=5.4 Hz, 1H), 4.15 (q, J=7.1 Hz, 2H), 3.46 (dd, J=11.0, 16.7 Hz, 1H), 3.29 (m, 1H), 3.10 (d, J=116.5, 2H), 2.35 (m, 1H), 2.16 (m, 1H), 1.23 (t, J=7.2 Hz, 3H).

Preparation of (2S-cis)-5-Benzyloxycarbonylamino-1,2,4,5,6,7-Hexahydro-4-Oxoazepino[3,2,1-hi]indole-2-Carboxylic Acid
To a solution of (2c-cis)-5-benzyloxycarbonylamino-1,2,4,5,6,7-hexahydro-4-oxoazepino[3,2,1-hi]indole-2-carboxylic acid, ethyl ester, (0.428 g, 1.05 mmol) in 1,4-dioxane (7.5 mL) and water (2.5 mL) was added 1M aqueous lithium hydroxide (1.6 mL, 1.6 mmol) and the resulting mixture was stirred at room temperature under nitrogen for 30 minutes. The reaction mixture was acidified to pH 3 with a 5% aqueous potassium bisulfate sodium chloride solution. The aqueous layer was back-extracted two times with ethyl acetate, and the combined organic layers were dried over sodium sulfate and evaporated to dryness to yield 0.395 g (99%) of title compound as a fine white solid; m.p.: 188-189xc2x0 C. TLC (methylene chloride-methanol-acetic acid, 9:1:1): Rf=0.55; 1H-NMR (300 MHz, CDCl3) xcex4 7.34-7.26 (m, 5H), 7.07-6.97 (m, 3H), 6.08 (d, J=5.7 Hz, 1H), 5.25 (dd, J=3.2, 9.8 Hz, 1H), 5.10 (s, 2H), 4.30 (m, 1H), 3.36 (m, 1H), 3.26 (m, 2H), 3.06 (d, J=12.0 Hz, 1H), 2.36 (m, 1H), 2.09 (m, 1H).

Preparation of (3R,S-cis)-6-Benzyloxycarbonylamino-5-oxo-2,3,4,5,6,7,8-hexahydro-1H-azepino[3,2,1-hi]quinoline-3-carboxylic acid, methyl ester
To a solution of (3R,S-cis)-6-Amino-5-oxo-2,3,4,5,6,7,8-hexahydro-1H-azepino[3,2,1-hi]quinoline-3-carboxylic acid, methyl ester (0.570 g, 2.1 mmol, prepared as described in Tetrahderon Letters 36, pp. 1593-1596 (1995) and U.S. Pat. No. 5,504,080 (Apr. 2, 1996)) in methylene chloride (6 mL) stirring at 0xc2x0 C. was added benzyl chloroformate (0.6 mL, 4.2 mmol) and triethylamine (1.2 mL, 8.4 mmol) and the resulting mixture was stirred under nitrogen for 30 minutes. The reaction was quenched with water then partitioned between ethyl acetate and 5% aqueous potassium bisulfate solution. The aqueous layer was back extracted two times with ethyl acetate, then the combined organic layers were washed with saturated sodium chloride solution, dried (sodium sulfate) and evaporated to dryness. Purification of the crude product by flash chromatography on silica gel (S/P brand silica gel 60 xc3x85, 230-400 mesh ASTM) eluting with ethyl acetate-hexane (2:1) gave 0.643 g (76%) of of the title compound as a white foam. TLC (methylene chloride-methanol, 95:5) Rf=0.8. 1H-NMR (300 MHz, CDCl3) xcex4 7.36-7.25 (m, 5H), 7.13-7.02 (m, 3H), 5.67 (d, J=7.8 Hz, 1H), 5.02 (t, J=9.15, 18.3 Hz, 2H), 4.34 (m, 1H), 3.70 (s, 3H), 3.16 (m, 1H), 2.69-2.56 (m, 5H), 2.06 (m, 1H). Mass spectrum: m/z 408 (M+H).

Preparation of (3R, S-cis)-6-Benzyloxycarbonylamino-5-oxo-2,3,4,5,6,7,8-hexahydro-1H-azepino [3,2,1-hi]quinoline-3-carboxylic acid
To a solution of (3R,S-cis)-6-Benzyloxycarbonylamino-5-oxo-2,3,4,5,6,7,8-hexahydro-1H-azepino[3,2,1-hi]quinoline-3-carboxylic acid, methyl ester (0.622 g, 1.53 mmol) in 1,4-dioxane (10.5 mL) and water (3.5 mL) was added 1M aqueous lithium hydroxide (2.3 mL, 2.3 mmol) and the resulting mixture was stirred at room temperature under nitrogen for 1 hour. The reaction mixture was acidified to ca. pH 2 with a 5% aqueous potassium bisulfate solution, then partitioned between ethyl acetate and saturated sodium chloride solution. The aqueous layer was back extracted two times with ethyl acetate, and the combined organic layers were dried (sodium sulfate) and evaporated to yield 0.670 g of the title compound. TLC (methylene chloride-methanol-acetic acid, 32:1:1) Rf=0.35. 1H-NMR (300 MHz, CDCl3) xcex4 7.38-7.28 (m, 5H), 7.13-7.04 (m, 3H), 5.72 (d, J=8.1 Hz, 1H), 5.03 (s, 2H), 4.35 (m, 1H), 3.77-3.67 (m, 5H), 3.10 (m, 1H), 2.72-2.52 (m, 5H), 2.07 (m, 1H), 1.70 (m, 1H).

Preparation of N-(Benzyloxycarbonyl)-L-(Nxe2x80x2-Methyl-Nxe2x80x2-Methoxy)aspartamide xcex2-(tert-Butyl Ester)
To a solution of N-(benzyloxycarbonyl)-L-aspartic acid-xcex2-(tert-butyl)ester (14.65 g, 45.3 mmol, Bachem) in CH2Cl2 (150 mL) at 0xc2x0 C. (ice bath) under a nitrogen atmosphere was added 1-hydroxybenzotriazole hydrate (7.29 g, 47.6 mmol, Aldrich) followed by 1-ethyl-3-(3xe2x80x2,3xe2x80x2-dimethyl-1xe2x80x2-aminopropyl)carbodiimide hydrochloride (9.55 g, 49.8 mmol, Sigma). After stirring at 0xc2x0 C. for 15 min., N,O-dimethylhydroxylamine hydrochloride (5.10 g, 52.3 mmol, Aldrich) and N-methylmorpholine (5.8 mL, 53 mmol, Aldrich) were added. The mixture was allowed to warm to room temperature over 3 hours then stirred at room temperature for 16 hours. The solution was concentrated under vacuum and the residue partitioned between ethyl acetate-5% KHSO4 (200 mL each). The organic phase was washed in turn with 5% KHSO4, saturated sodium bicarbonate and saturated sodium chloride solutions; dried over anhydrous sodium sulfate and evaporated to an oil. The oil was crystallized from hexane to give the title product (16.10 g, 97% yield) as a fluffy white crystalline solid. TLC (ethyl acetate), single spot (UV and PMA): Rf=0.37.
A similar procedure to the one above, starting with 29.3 g of N-(benzyloxycarbonyl)-L-aspartic acid-xcex2-(tert-butyl)ester (2-fold scale up) gave 31.18 g (94% yield) of the title product.

Preparation of N-(Benzyloxycarbonyl)-L-Aspartic Acid Semicarbazone xcex2-(tert-Butyl)Ester
To a solution of N-(benzyloxycarbonyl)-L-(Nxe2x80x2-methyl-Nxe2x80x2-methoxy)aspartamide xcex2-(tert-butyl ester) (15.50 g, 42.3 mmol) in anhydrous ether (400 mL) at 0xc2x0 C. (ice bath) under a nitrogen atmosphere was added dropwise to a 1.0 M solution of LiAlH4 in ether (22.0 mL, 22.0 mmol, Aldrich) at such a rate as to keep the reaction solution temperature between 0-5xc2x0 C. (addition time 15-20 min). After the addition of the lithium aluminum hydride reagent was complete, the mixture was stirred at 0-5xc2x0 C. for 1 hr, then quenched by the dropwise addition of 0.3 N KHSO4 solution (100 mL). The resultant mixture was transferred to a separatory funnel adding sufficient 5% KHSO4 solution (75 mL) to dissolve the solids. The organic phase was separated and the combined aqueous washes back-extracted with ether (100 mL). The combined ether extracts were washed with saturated NaCl solution, dried over anhydrous sodium sulfate and concentrated in vacuo with minimal heating. TLC (ethyl acetate): streaky spot (UV and PMA) Rf=0.48. TLC (methanol/methylene chloride, 1:9) major spot (UV and PMA): Rf=0.75.
The crude aldehyde was immediately taken up in aqueous ethanol (45 mL water/105 mL alcohol), placed in an ice bath and treated with sodium acetate (3.82 g, 46.6 mmol) and semicarbazide hydrochloride (5.20 g, 46.6 mmol, Aldrich). The mixture was stirred at 0xc2x0 C. (ice bath) under a nitrogen atmosphere for 3 hrs, allowed to warm to room temperature, and stirred overnight (16 hrs). Most of the ethanol was removed under vacuum and the residue partitioned between ethyl acetate and water (100 mL each). The organic phase was washed sequentially with 5% KHSO4, saturated sodium bicarbonate and saturated sodium chloride solutions; dried over anhydrous sodium sulfate and evaporated to dryness. The crude product of this reaction was combined with that of two similar procedures starting with 15.40 g and 4.625 g of N-(benzyloxycarbonyl)-L-(Nxe2x80x2-methyl-Nxe2x80x2-methoxy)aspartamide xcex2-(tert-butyl ester) (total: 35.525 g, 97 mmol) and these combined products were purified by flash chromotagraphy on silica gel eluting with acetone/methylene chloride (3:7) then methanol-acetone-methylene chloride (0.5:3:7) to give pure title product (27.73 g, 78.5%) as a colorless foam. TLC (MeOHxe2x80x94CH2Cl2, 1:9): single spot (UV and PMA), Rf=0.51.

Preparation of L-Aspartic Acid Semicarbazone xcex2-(tert-Butyl) Ester, p-Toluenesulfonate Salt
To a solution of N-(benzyloxycarbonyl)-L-aspartic acid semicarbazone P-(tert-butyl)ester (13.84 g, 38.0 mmol) in absolute ethanol (250 mL) was added 10% Pd/C (1.50 g, Aldrich) and the resulting mixture stirred under an atmosphere of hydrogen (balloon) until TLC (methanol/methylene chloride, 1:9) indicated complete consumption of the starting material (60 min). Note: It is important to follow this reaction closely since the product can be over-reduced. The mixture was filtered though Celite and evaporated to an oil. The oil was chased with methylene chloride (2xc3x9775 mL) then with methylene chloride/toluene (1:1, 75 mL) to give the crude amine as a white crystalline solid. TLC (EtOAc-pyridine-AcOH-H2O; 60:20:5:10) single spot (UV and PMA) Rf=0.24. Note: In this TLC system, any over-reduced product will show up immediately below the desired product, Rf=0.18 (PMA only).
The crude amine was taken up in CH3CN (60 mL) and treated with a solution of p-toluenesulfonic acid monohydrate (7.22 g, 38.0 mmol) in acetonitrile (60 mL). The crystalline precipitate was collected, washed with acetonitrile and ether, and air-dried to give the title compound (13.95 g, 92% yield) as a white, crystalline solid.
The optical purity of this material was checked by conversion to the corresponding Mosher amide [1.05 equiv (R)-(xe2x88x92)-xcex1-methoxy-xcex1-(trifluoromethyl)phenylacetyl chloride, 2.1 equivalents of i-Pr2NEt in CH2Cl2, room temperature, 30 min]. The desired product has a doublet at 7.13 ppm (1H, d, J=2.4 Hz, CHxe2x95x90N) while the corresponding signal for its diastereomer is at 7.07 ppm. The optical purity of the title compound obtained from the above procedure is typically  greater than 95:5.
Assay for Inhibition of ICE/ced-3 Protease Family Activity
A. Determination of IC50 Values
Fluorescence enzyme assays detecting the activity of the compounds of Formula 1 utilizing the recombinant ICE and CPP32 enzymes were performed essentially according to Thornberry et al. (Nature, 356:768:774 (1992)) and Nicholson et al. (Nature, 376:37-43 (1995)) respectively, (herein incorporated by reference) in 96 well microtiter plates. The substrate is Acetyl-Tyr-Val-Ala-Asp-amino-4-methylcoumarin (AMC) for the ICE assay and Acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin for the CPP32, Mch2, Mch3 and Mch5 assays. Enzyme reactions were run in ICE buffer (25 mM HEPES, 1 mM EDTA, 0.1% CHAPS, 10% sucrose, pH 7.5) containing 2 mM DTT at room temperature in duplicate. The assays were performed by mixing the following components:
50 xcexcL ICE, Mch2, Mch5, CPP32 (18.8, 38, 8.1 and 0.153 nM concentrations, respectively) or Mch3 (1 unit) enzyme in ICE buffer containing either 8.0 (ICE, Mch2, Mch3, CPP32) or 20 (Mch5) mM DTT;
50 xcexcL compound of Formula 1 or ICE buffer (control); and
100 xcexcL of 20 xcexcM substrate.
The enzyme and the compound of Formula 1 to be assayed were allowed to preincubate in the microtitre plate wells for 30 minutes at room temperature prior to the addition of substrate to initiate the reaction. Fluorescent AMC product formation was monitored for one hour at room temperature by measuring the fluorescence emission at 460 nm using an excitation wavelength of 360 nm. The fluorescence change in duplicate (control) wells were averaged and the mean values were plotted as a function of inhibitor concentration to determine the inhibitor concentration producing 50% inhibition (IC50). The results of this assay are set forth below in Table 1.
The reference compound for this assay was Cbz-ValAlaAsp-H and the values are denoted in Table 1 as xe2x80x9cReferencexe2x80x9d.
B. Determination of the dissociation constant Ki and irreversible rate constant k3 for irreversible inhibitors
For the irreversible inhibition of a ICE/ced-3 Family Protease enzyme with a competitive irreversible inhibitor; using the model represented by the following formulas: 
The product formation at time t may be expressed as:                                           [            P            ]                    t                =                                            [              E              ]                        T                    ⁢                      (                                                            [                  S                  ]                                ⁢                                  K                  i                                                                                                  [                    I                    ]                                    ⁢                                      K                    s                                                  ⁢                                  xe2x80x83                                                      )                    ⁢                      xe2x80x83                    ⁢                                    (                                                k                  s                                                  k                  3                                            )                        ⁢                          xe2x80x83                        [                          1              -                              ⅇ                                                      -                                          k                      3                                                        ⁢                                      t                    /                                          (                                              1                        +                                                                                                            K                              i                                                                                      [                              I                              ]                                                                                ⁢                                                      (                                                          1                              +                                                                                                [                                  S                                  ]                                                                                                  K                                  s                                                                                                                      )                                                                                              )                                                                                            ]                                              Equation  1            
where E, I, EI and E-I denote the active enzyme, inhibitor, non-covalent enzyme-inhibitor complex and covalent enzyme-inhibitor adduct, respectively. The Ki value is the overall dissociation constant of the reversible binding steps, and k3 is the irreversible rate constant. The [S] and Ks values are the substate concentration and dissociation constant of the substrate bound to the enzyme, respectively. [E]T is the total enzyme concentration.
The above equations were used to determine the Ki and k3 values of a given inhibitor bound to a ICE/ced-3 family protease. Thus, a continuous assay was run for sixty minutes at various concentrations of the inhibitor and the substrate. The assay was formulated essentially the same as described above for generating the data in Table 1, except that the reaction was initiated by adding the enzyme to the substrate-inhibitor mixture. The Ki and k3 values were obtained by simulating the product AMC formation as a function of time according to Equation I. The results of this second assay are set forth below in Table 2.
The reference compound for this assay was Cbz-ValAlaAsp-CH2F and the values are denoted in Table 2 as xe2x80x9cReferencexe2x80x9d.